Baseline data collections of lipopolysaccharide content in 414 herbal extracts and its role in innate immune activation

Some herbal extracts contain relatively high amounts of lipopolysaccharide (LPS). Because orally administered LPS activates innate immunity without inducing inflammation, it plays a role as an active ingredient in herbal extracts. However, the LPS content in herbal extracts remains extensively unevaluated. This study aimed to create a database of LPS content in herbal extracts; therefore, the LPS content of 414 herbal extracts was measured and the macrophage activation potential was evaluated. The LPS content of these hot water extracts was determined using the kinetic–turbidimetric method. The LPS concentration ranged from a few ng/g to hundreds of μg/g (Standard Escherichia coli LPS equivalent). Twelve samples had a high-LPS-content of > 100 μg/g, including seven samples from roots and three samples from leaves of the herbal extracts. These samples showed high phagocytosis and NO production capacity, and further investigation using polymyxin B, an LPS inhibitor, significantly inhibited macrophage activation. This study suggests that some herbal extracts contain sufficient LPS concentration to activate innate immunity. Therefore, a new approach to evaluate the efficacy of herbal extracts based on their LPS content was proposed. A database listing the LPS content of different herbal extracts is essential for this approach.

functional properties.Additionally, Pantoea agglomerans was isolated as the dominant LPS symbiont in wheat 33 .Oral consumption of Pantoea agglomerans LPS (LPSp) enhanced phagocytosis of abdominal macrophages in mice, but this effect was not observed in TLR4-deficient mice 34 .This indicates that orally administered LPS promotes foreign body removal via innate immunity using TLR4.Furthermore, in disease prevention and treatment experiments, oral LPSp administration was found to enhance the effect of anticancer drugs 35 , promote the treatment of lung metastases 36 , inhibit itching in atopic dermatitis 25 , prevent atherosclerosis in apolipoprotein-E (ApoE)-deficient mice 37 , prevent dementia in brain diabetes-induced mice 38 etc.Additionally, a recent study reported that orally administered LPS suppressed diabetic symptoms by increasing the expression of insulin signaling-related factors, especially adiponectin, in adipose tissue in type 2 diabetes mellitus, a disease supposedly LPS-induced 39 .Furthermore, LPSp has been confirmed to be highly safe in rats, with no adverse effects after oral administration at 2 g/kg body weight (BW) or higher 40 .
From the above-mentioned studies, LPS from ingested food is likely to activate and regulate innate immunity.Furthermore, considering its presence in herbal extracts, there is a possibility that the consumption of herbal extracts may activate the body innate immunity regulation.Herbal extracts are defined as naturally occurring unrefined substances from any part or parts of plants, animals, and other organisms with one or more active ingredients intended to alleviate, treat, or prevent diseases 41 .The above-mentioned wheat is a herbal extract listed in the "The Japanese standards for nonpharmacopoeial herbal extracts 2022" and is called Shobaku 42 .The overall health benefits of consuming herbal extracts are generally thought to be due to the low molecular weight of the active ingredient.However, a sufficient amount of LPS in the herbal extracts can activate the innate immune system; therefore, LPS should also be considered an active ingredient of herbal extracts.As the innate immune system-activating effect of orally administered LPS is coming to light 34 , LPS in herbal extracts as a component of the effects of Chinese herbal medicine deserves attention.Thus, a database of the LPS content in herbal extracts and food ingredients is required to make this concept common knowledge.
In 1992, our group screened approximately 60 plant samples, including herbal extracts, for their LPS content and found that some plants had a high LPS content of over 100 μg/g 32 .However, since then, little effort has been made to measure the LPS content in herbal extracts.Montenegro et al. was the first to report on LPS's ability to activate macrophages, an innate immunity mechanism, in Kampo medicine 43 .In this study, they showed that the macrophage-activating component of Juzen-taiho-to, an immune-boosting Kampo medicine formulated from 10 herbal extracts, is correlated with the amount of LPS, which is obtained from symbiotic bacteria existing in one of its ingredients.Their study showed that LPS is a functional component that activates and controls macrophages (innate immunity) in Juzen-taiho-to; hence, LPS can be regarded as an active component of the innate immune system of numerous herbal extracts because most herbal extracts have symbiotic bacteria that supply LPS.Therefore, if information on the LPS content found in herbal extracts can be obtained, the knowledge that oral intake of LPS does not induce inflammation can be enforced, and a new perspective on the concept of LPS as an effective component of herbal extracts can be provided.However, data evaluating herbal extracts from the LPS viewpoint are currently extremely limited, as described above.
Thus, to provide a comprehensive list of the LPS content of herbal extracts and other food ingredients, the LPS content of 414 herbal extracts were measured and compared.Additionally, the macrophage activation potential of herbal extracts with particularly high-LPS-content was compared and measured to investigate the connection between LPS content and macrophage activity.

Measurement of the LPS content of herbal extracts
By measuring Limulus activity, the amount of LPS in the herbal extracts was examined.The LPS concentrations of 414 samples of herbal extracts obtained from vascular plants, fungi, and others ranging from below the detection limit to several 100 μg/g are shown in Table 1. Figure 1 shows the distributions of the LPS concentrations within each species.Herbal extracts from vascular plants were further divided according to their parts.For this analysis, the groups were classified according to the crude drug classification method.The results showed that herbal extract ingredients with high LPS contents were mostly found in the vascular plant group.Comparisons between vascular plant parts indicated that roots (107 samples) had significantly higher LPS levels than fruits (69 samples) and seeds (22 samples), and leaves (68 samples) had significantly higher LPS levels than fruits (69 samples).The average LPS concentration in all samples was 17.4 ± 69.3 μg/g.There are 12 samples containing high LPS concentration > 100 μg/g, 80 samples containing concentrations of 10-100 μg/g, and 162 samples containing concentrations of 1-10 ng/g.The 12 samples with significantly high LPS contents, which are listed in Table 2, were selected to further test the macrophage-activating effect of LPS.The measured LPS content indicated that herbal extracts contain LPS and that the amount of LPS in each plant's part varies depending on the parts from which they are derived.

Measurement of the macrophage activation potential of the herbal extracts
Twelve herbal extract samples with LPS levels of ≥ 100 μg/g were tested for macrophage activation potential.Macrophage activation potential was assessed by measuring phagocytosis and nitric oxide (NO) production by stimulating RAW 264.7 cells with the herbal extracts.Stimulation using purified LPSp served as a positive control.Phagocytic activity was increased in all samples compared with that in the non-stimulated control group (Fig. 2).The phagocytosis ability of RAW 264.7 cells was increased when stimulated with Oat (Avena sativa L.), Sacred lotus (Nelumbo nucifera Gaertn.),Aralia rhizome (Aralia cordata Thunb.),Fortune's drynaria rhizome (Drynaria roosii Nakaike), Couch grass (Elytrigia repens (L.) Gould), Angelica dahurica root (Angelica dahurica), Common ducksmeat (Spirodela polyrhiza (L.) Schleid.),Corn silk (Zea mays L.), and Bupleurum root (Bupleurum falcatum L.) compared with the positive control LPSp.

Discussion
Herbal extracts have several health-benefiting effects, such as hemostatic 44,45 , antifebrile 46,47 , detoxifying 48 , sweating 49 , and immunostimulating effects 50 , most of which are low molecular weight substances and have significantly contributed to the development of pharmaceuticals as the beginning of numerous medicines.LPS in herbal extracts supposedly causes this immunostimulating effect because previous LPS screening study revealed that some herbal extracts contain high LPS amounts (> 100 μg/g) 32 and previous studies have shown that the oral intake of LPS enhances immunity and effectively prevents and improves various diseases, including cancer, viral infection, atopic dermatitis, diabetes, atherosclerosis, and Alzheimer's disease 38,[51][52][53] .Although there are more than several hundred herbal extracts worldwide and the possibility that the LPS in these herbal extracts playing a role in their functions is high, the LPS amount in them has never been measured or compared among the parts of plants from which they were obtained.Therefore, this study aimed to create a database of LPS levels in herbal extracts by measuring LPS levels in over 400 herbal extract samples stored at the Faculty of Pharmaceutical Sciences, Hokkaido University of Science, and to provide a basis for research to assess the immunostimulatory effects of herbal extracts and LPS's contribution to these effects.
Table 1 shows the amount of LPS in 414 herbal extracts.LPS concentrations were widely distributed from a few μg/g to several hundred μg/g (Fig. 1).LPS content was shown to be significantly higher in roots (107 samples) than in fruits (69 samples) or seeds (22 samples) in terms of LPS concentration.Of the 414 herbal extracts measured in this study, approximately 100 herbal extracts contained ≥ 10 μg/g of LPS.Twelve of the herbal extracts exhibited very high LPS levels of over 100 μg/g.Comparison among vascular plant parts showed that the overall LPS level in root-derived herbal extracts was high and significantly higher than that in seed-and fruit-derived herbal extracts.Over half (seven) of the 12 high-LPS-content herbal extracts were root-derived.Most vascular plants are symbiotic with soil bacteria in their roots [54][55][56] .Symbiotic bacteria in soil promote plant growth through  www.nature.com/scientificreports/their involvement in nitrogen fixation, nutrient supply, and disease defense.Such bacteria are called plant growthpromoting rhizospheric microorganisms (PGPR) 57 ; among them, bacteria of the genera Pseudomonas, Azospirillum, Bradyrhizobium, and Rhizobium are particularly essential.These bacteria are gram-negative bacteria and, therefore, may contribute to the high-LPS-content in the roots of herbal extracts.Montenegro et al. reported that 519 genera of bacteria are found in Angelica sinensi, a root-derived herbal extract that constitutes Juzen Daihoto, a Chinese herbal medicine known for its immunostimulating properties 43 .Among them, Rahnella, a gram-negative bacterium found in soil and fresh water, is abundant in Angelica sinensi.It was stipulated that the LPS content in Angelica sinensi is involved in the immunity-enhancing effects of Juzen Daihoto.The LPS content of Angelica sinensi (also called Angelica acutiloba Kitag. in Japan) was also measured in this study and it was shown that it contained 16 μg/g LPS, the 61st highest LPS content among all 414 samples in Table 1 (herb sample no.202).These results suggest that the LPS amount in the root-derived herbal extract correlates with the number of soil-derived microorganisms that symbiotically coexist with the root-derived microorganisms during growth.These microorganisms are mostly gram-negative bacteria that contain a high LPS amount.On the other hand, the variation within each part group is large, suggesting that the high or low LPS content may not so much dependent on the part of the sample.The amount of LPS contained in plants is considered to be derived from symbiotic bacteria.Therefore, the type and amount of symbiotic bacteria may vary depending on the origin of the plant, time of collection, variety, and cultivation method.Consequently, it is meaningful to measure multiple samples, but it is difficult to obtain multiple lots of crude drugs because most of them are imported.Therefore, we decided to use the variation in LPS content of one crude drug, brown rice, as a model for the variation in a single crude drug sample.In a previous study, we obtained brown rice from 15 different locations in Japan and measured LPS content in the 10.9 ± 4.3 μg/g range 58 .Although the LPS content of brown rice may not necessarily be universalizable to other crude drugs, we believe that this can be used as a reference value for the degree of variation in LPS content.The range of LPS content in this one sample was relatively stable compared to the range of 0.001-100 μg/g in the LPS content data (Table 1, Fig. 1) obtained for individual crude drugs.Therefore, based on this fact, we conducted the experiment with the belief that the approximate degree of LPS content could be evaluated with a single sample.
In this study, Limulus amebocyte lysate (LAL) test was used to detect LPS in the herbal extracts.However, it has been reported that β-1,3-glucan also reacts with LAL, so, there is a possibility of measuring plant-derived β-1,3-glucan contaminant with ordinary LAL.In this study, this contamination is prevented by using an LAL test kit containing a carboxymethylated curdlan which has reported act as a blocker of β-1,3-glucan mediated coagulation pathway 59 .Therefore, the limulus activity detected in this study were specific to LPS.
The macrophage-activating ability of LPS is a fundamental LPS action 34 .Therefore, the macrophage activation potential of herbal extracts by phagocytosis and NO production was assessed using macrophage-like RAW 264.7 cells.RAW246.7 cells transduce LPS signaling via TLR4 60 .In addition, many mammalian innate immune system cells, including humans, express TLR4 61 .Therefore, even though this study used mouse macrophage cells as a representative model, it is safe to assume that LPS contained in crude drugs is functional for mammals in general, including humans.However, further research is needed to determine the effects of LPS in humans,  2. The concentrations of herbs and LPSp added were adjusted so that the LPS concentration was 100 ng/ml.The dotted line represents the phagocytosis percentage of RAW 264.7 cells without any external stimulation (medium only).Each bar represents the mean of two independent measurements, and the error bars represent the standard deviation.especially when administered orally.Twelve samples containing particularly high amounts of LPS (100 μg/g) were examined using these methods.The results showed that herbal extracts increased the phagocytosis capability of RAW 264.7 cells (Fig. 2).The NO production by RAW 264.7 cells caused by these samples was found to be higher, similar, or lower than purified LPSp, depending on the 12 herbal extracts (Fig. 3).The LPS itself in the group that exhibited higher activity may display high macrophage activation.However, it is speculated that a synergistic effect with macrophage activators, such as bacterial-derived nucleic acids, peptidoglycans, and flagellin, may be observed.Conversely, those that exhibited weaker activity than LPSp derived from Enterobacteriaceae may be because of the nature of the symbiotic gram-negative bacteria, as some LPSs, such as Bacteroides, are weak in biological activity, which depends on their lipid A structure 62,63 .Additionally, NO production was significantly (> 70%) reduced in all RAW 264.7 cells stimulated with 12 herbal extracts when polymyxin B, an LPS inhibitor, was added (Fig. 4).These results suggest that LPS is responsible for most of the macrophage activation potential of herbal extracts.However, the strength of the macrophage-activating ability of the herbal extracts is not  2.The amount of LPS needed to induce 5 µM more nitrite than LPSp used as control is (a) less than LPSp, (b) equivalent to LPSp, and (c) more than LPSp in this group.The dotted lines represent 5 µM Nitrite.The trendline equations (dashed lines) and R 2 of each line are listed in Table 3.
proportional to the amount of LPS contained and may significantly differ among various symbiotic bacteria.Therefore, in studying the innate immune activation potential of herbal extracts, it is necessary to assess and clarify their unique qualities.Herbal extracts are often prescribed in daily doses of 1-10 g 64,65 .Of the 414 herbal extracts for which LPS levels were measured in this study, 98 contained over 10 µg/g LPS, and oral intake of LPS increased the phagocytic activity of abdominal macrophages in mice at 10 µg/kg BW for 7 days 34 , induced increase in capillary vascularity at 10 µg/kg BW in human randomized control trial studies 66 , and in fish, 5-20 μg/kg BW increased the ability to prevent infection 67 .Based on these studies, 10 μg/kg BW of LPS can activate innate immunity, which is 500 μg/ day for a 50 kg human.Therefore, consuming a daily dose of herbal extracts may mean taking in an effective amount of LPS, meaning that LPS may contribute to the medicinal effects of the herbal extracts.Juzen Daiho-to, a combination of herbal extracts, reportedly has preventive and ameliorative effects against diabetes and cancer partly because LPS is one of its ingredients 68,69 .The 414 herbal extract samples measured in this study are much greater than the 157 listed in the Japanese Pharmacopoeia.These should be sufficient populations for primary screening based on the efficacy of oral LPS intake over immune functions and the activation of immune cells using macrophages and other cells in herbal extracts.However, because the LPS content of plants is obtained from the symbiotic gram-negative bacterial population and may differ greatly depending on the time of collection, variety, cultivation method, etc., the LPS content of the samples to be studied should be analyzed with caution on a sample-by-sample basis.

Sample preparation
All dried samples were purchased from Tochimoto Tenkaido Co., Ltd.(Osaka, Japan).The dried samples were pulverized, and 100 mg powdered samples were extracted in 1 ml distilled water for 20 min at 90 °C.Subsequently, the samples were sonicated for 20 min and vortexed for two minutes to extract LPS.Next, the supernatants were obtained after centrifugation at 830 × g for 15 min.All methods involving the dried samples were carried out in accordance with relevant guidelines 70 .

Measurement of the LPS contents of herbal extracts
The LPS concentration in the samples were assayed using the kinetic-turbidimetric method.All samples were diluted 10,000-fold using pyrogen-free distilled water.Sample supernatants (0.2 ml) were added to LAL-ES in a glass tube (Limulus ES-II single test; Wako Pure Chemical Industries Ltd., Osaka, Japan).After a few seconds of votexing, the gelation time was measured using a Toxinometer ET-6000 (Wako Pure Chemical Industries Ltd.), and the specific activity was calculated using an LS Toximaster (Wako Pure Chemical Industries Ltd.), a data acquisition program for the Toxinometer.
The LAL test kits of Wako contain carboxymethylated curdlan in freeze-dried reagents, which stops β-dglucans from triggering an interference in the test.Therefore, this test kit used in this study is specific to LPS 59 .

Phagocytosis assay
Phagocytic activity was measured using flow cytometry as previously described with minor modifications 71 .Briefly, the mouse macrophage/monocyte cell line RAW 264.7 cells (obtained from TIB-71; ATCC, Manassas, VA, USA) were treated for 18 h with extracts in a 48-well plate.The extract concentrations were measured so that the LPS content was 100 ng/ml.Next, fluorescent latex beads (Fluoresbrite ® YG Microspheres 1.0 μm; Polysciences, Warrington, PA) at a cell: bead ratio of 1:10 were added and incubated for one hour.Cells were washed to eliminate non-internalized particles and detached from the well plate with 0.25% trypsin treatment (Life Technologies, Carlsbad, CA, USA).The phagocytosis rate of the cells was measured using a Beckman Coulter Gallios flow cytometer and Kaluza software (Beckman Coulter, Indianapolis, IN).

Nitric oxide (NO) production by murine macrophages
In a 48-well plate, cells from the mouse macrophage/monocyte cell line RAW 264.7 were plated at 8 × 105 cells/ ml and treated with herbal extracts.The added extract concentrations were measured, so that the LPS content was 1, 10, and 100 ng/ml.The plate was incubated at 37 °C and 5% CO 2 .After 24-h incubation with extracts, the supernatants were collected, and the concentrations of nitrite (NO 2− ) released into the culture media were measured using Griess reagent.In addition, 100 μl Griess reagent was added to 100 μl diluted culture media in the wells of microtiter plates.After incubation at room temperature for ten minutes, absorbance at 570 nm was determined using an automated microplate reader (BIO-RAD, Hercules, CA, USA).The NO assay was conducted in duplicate.To determine the percentage of NO produced by the LPS in the herbal extracts, the concentrations of the extracts were measured, so that the LPS content was 10 ng/ml, and polymyxin B (Sigma-Aldrich, St. Louis, MO, USA) was added to each culture at a final concentration of 10 μg/ml.

Statistical analysis
Data are presented as mean ± standard deviation (SD).Statistical analyses (Steel-Dwass test and Pearsons' correlation) were performed using the JMP statistical software, version 17. 0. 0 (SAS Institute Inc., Cary, NC, USA).Statistical differences between multiple groups in the box-and-whisker plot were calculated using the Steel-Dwass test.A p-value < 0.05 was considered statistically significant.The line equation and its R 2 value in Table 3 were performed using Microsoft Excel.

Figure 1 .
Figure 1.The distribution of the LPS concentration of the 414 samples measured using the Limulus reaction.The samples were divided into plants, fungi, and others.The plant samples were further categorized according to their parts.*p-value < 0.05 for Steel-Dwass test.

Figure 2 .
Figure 2. The percentage of phagocytic activity of RAW 264.7 cells stimulated by the 12 herbal extract samples containing the highest LPS levels are listed in Table2.The concentrations of herbs and LPSp added were adjusted so that the LPS concentration was 100 ng/ml.The dotted line represents the phagocytosis percentage of RAW 264.7 cells without any external stimulation (medium only).Each bar represents the mean of two independent measurements, and the error bars represent the standard deviation.

Figure 3 .
Figure 3. Dose-response curve of macrophage activation capacity determined by measuring the amount of NO produced as the amount of nitrite produced by RAW 264.7 cells stimulated by adding 1, 10, and 100 ng/ ml per LPS to the 12 herbal extract samples containing the highest LPS content listed in Table2.The amount of LPS needed to induce 5 µM more nitrite than LPSp used as control is (a) less than LPSp, (b) equivalent to LPSp, and (c) more than LPSp in this group.The dotted lines represent 5 µM Nitrite.The trendline equations (dashed lines) and R 2 of each line are listed in Table3.

Table 1 .
The phagocytosis ability of RAW 264.7 cells with Ginger Plant LPS concentrations of 414 samples of herbal extracts.For herbal extracts with multiple scientific names, the scientific names listed in this table are those most used in Japan.

Table 2 .
Twelve herbal extract samples with significantly higher LPS content than the other samples.The samples are listed in the order of high concentration.

Table 3 .
The equivalent amount of herbal extracts per LPS content needed to induce 5 µM nitrite, which is the relative nitrite induction strength compared with LPSp.The trendline equations and R 2 of each line in Fig.3are also listed.Macrophage activation potential determined by measuring the NO production of RAW 264.7 cells stimulated by the 12 herbal samples containing the highest LPS levels listed in Table2.The percentage of NO produced by RAW 264.7 cells stimulated by LPS content (black bars) and other components (white) in the herbal extract samples.The concentrations of herbs and LPSp added were adjusted, making the LPS content 10 ng/ml.The black area represents the percentage of induced NO 2 being decreased following polymyxin B addition, representing the percentage of NO 2 induced by the LPS content in the herb samples.Each bar represents the mean of two independent measurements, and the error bars represent the standard deviation.